Hiv vaccine and method of use

ABSTRACT

This invention is directed to pharmaceutical compositions comprising an HIV antigen and a mucosal adjuvant and methods for raising an immune response in a subject by administering these compositions. Preferably, the pharmaceutical compositions of the invention can be used to treat or prevent HIV infection.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to pharmaceutical compositions comprising anHIV antigen and a mucosal adjuvant and methods for raising an immuneresponse in a subject by administering these compositions. Preferably,the pharmaceutical compositions of the invention can be used to treat orprevent HIV infection.

HIV antigens suitable for use in this invention include envelopeproteins such as gp120 and gp160 proteins, and antigenic fragments andderivatives thereof, such as oligomeric gp140 (Ogp140). Preferably, theantigens of the invention are optimized for immunogenicity.

The pharmaceutical compositions of this invention are suitable formucosal delivery, preferably intranasal, intra-vaginal and intra-rectaldelivery. Mucosal adjuvants suitable for use in this invention includedetoxified mutants of E. coli heat labile toxin (LT), such as LTR72 andLTK63.

2. State of the Art

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modem medicine and worldwide sexualtransmission of HIV, is the leading cause of AIDS. There are, as yet, nocures or vaccines for AIDS. Therefore, construction of a vaccine or drugthat can specifically protect against sexual transmission at the site ofentry is highly desirable.

In 1983-1984, three groups independently identified the suspectedetiological agent of AIDS. See, e.g., Barre-Sinoussi et al. (1983)Science 220:868-871; Montagnier et al., in Human T-Cell Leukemia Viruses(Gallo, Essex & Gross, eds., 1984); Vilmer et al. (1984) The Lancet1:753; Popovic et al. (1984) Science 224:497-500; Levy et al. (1984)Science 225:840-842. These isolates were variously calledlymphadenopathy-associated virus (LAV), human T-cell lymphotropic virustype III (HTLV-III), or AIDS-associated retrovirus (ARV). All of theseisolates are strains of the same virus, and were later collectivelynamed Human Inmunuodeficiency Virus (HIV). With the isolation of arelated AIDS-causing virus, the strains originally called HIV are nowtermed HIV-1 and the related virus is called HIV-2 See, e.g., Guyader etal. (1987) Nature 326:662-669; Brun-Vezinet et al. (1986) Science233:343-346; Clavel et al. (1986) Nature 324:691-695. Consequently,there is a need in the art for compositions and methods suitable fortreating and/or preventing HIV infection worldwide.

Although there is some discrepancy as to the effectiveness ofcell-mediated or antibody-mediated responses in protection againstdisease, there is general consensus that generation of bothcell-mediated and antibody-mediated responses is highly desirable.Antibody mediated responses would inhibit binding of the virus to itstargets in vaginal or rectal tissues, i.e., at the site of transmission,whereas cell-mediated responses would play a role in the eradication ofinfected cells.

Thus, as most HIV infections are transmitted through the female genitaltract followed by systemic spread of the virus, induction of local aswell as systemic immunity is greatly sought.

BRIEF SUMMARY OF THE INVENTION

This invention is directed to pharmaceutical compositions comprising anHIV antigen and a mucosal adjuvant and methods for raising an immuneresponse in a subject by administering these compositions. Preferably,the pharmaceutical compositions of the invention can be used to treat orprevent HIV infection.

HIV antigens suitable for use in this invention include envelopeproteins such as gp120 and gp 160 protein, and antigenic fragments andderivatives thereof, such as oligomeric gp140 (Ogp140). Preferably, theantigens of the invention are optimized for immunogenicity.

The pharmaceutical compositions of this invention are suitable formucosal delivery, preferably intranasal, intra-vaginal and intra-rectaldelivery. Mucosal adjuvants suitable for use in this invention includedetoxified mutants of E. coli heat labile toxin (LT), such as LTR72 andLTK63.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to pharmaceutical compositions comprising anHIV antigen and a mucosal adjuvant and methods for raising an immuneresponse in a subject by administering these compositions. Thepharmaceutical compositions of this invention are suitable for mucosaldelivery, preferably intranasal, intra-vaginal and intra-rectaldelivery. Mucosal adjuvants suitable for use in this invention includedetoxified mutants of E. coli heat labile toxin (LT), such as LTR72 andLTK63. In addition, the compositions of this invention can be used incombinations of mucosal prime/systemic boost or systemic prime/mucosalboost.

In order to facilitate an understanding of the invention, selected termsused in the application will be discussed below.

The term “polynucleotide”, as known in the art, generally refers to anucleic acid molecule. A “polynucleotide” can include both double- andsingle-stranded sequences and refers to, but is not limited to, cDNAfrom viral, prokaryotic or eukaryotic MRNA, genomic RNA and DNAsequences from viral (e.g. RNA and DNA viruses and retroviruses) orprokaryotic DNA, and especially synthetic DNA sequences. The term alsocaptures sequences that include any of the known base analogs of DNA andRNA, and includes modifications such as deletions, additions andsubstitutions (generally conservative in nature), to the nativesequence, so long as the nucleic acid molecule encodes a therapeutic orantigenic protein. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts that produce the antigens. Modifications ofpolynucleotides may have any number of effects including, for example,facilitating expression of the polypeptide product in a host cell.

The polynucleotides used in the present invention includepolynucleotides encoding for an immunogenic fragment or derivativethereof. Such immunogenic fragments or derivatives thereof includefragments encoding for a B-cell epitope or a T-cell epitope as discussedbelow.

As used herein, the terms “polypeptide” and “protein” refer to a polymerof amino acid residues and are not limited to a minimum length of theproduct. Thus, peptides, oligopeptides, dimers, multimers, and the like,are included within the definition. Both full-length proteins andfragments thereof are encompassed by the definition. The terms alsoinclude postexpression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein that includes modifications, such as deletions, additions andsubstitutions (generally conservative in nature), to the nativesequence, so long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts that producethe proteins or errors due to PCR amplification.

By “isolated” is meant, when referring to a polynucleotide or apolypeptide, that the indicated molecule is separate and discrete fromthe whole organism with which the molecule is found in nature or, whenthe polynucleotide or polypeptide is not found in nature, issufficiently free of other biological macromolecules so that thepolynucleotide or polypeptide can be used for its intended purpose.

The phrase “antigen”, as used herein, refers to a molecule containingone or more epitopes (either linear, conformational or both) that willstimulate a host's immune system to make a humoral and/or cellularantigen-specific response. The term is used interchangeably with theterm “immunogen.” Normally, a B-cell epitope will include at least about5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope,such as a CTL epitope, will include at least about 7-9 amino acids, anda helper T-cell epitope at least about 12-20 amino acids. Normally, anepitope will include between about 7 and 15 amino acids, such as, 9, 10,12 or 15 amino acids. The term “antigen” denotes both subunit antigens,(i.e., antigens which are separate and discrete from a whole organismwith which the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes.

Furthermore, for purposes of the present invention, an “antigen” refersto a protein that includes modifications, such as deletions, additionsand substitutions (generally conservative in nature), to the nativesequence, so long as the protein maintains the ability to elicit animmunological response, as defined herein. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts that produce the antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, specific effectorcells, such as B and plasma cells as well as cytotoxic T cells, againstcells displaying peptide antigens in association with MHC molecules ontheir surface. A “cellular immune response” also refers to theproduction of cytokines, chemokines and other such molecules produced byactivated T-cells and/or other white blood cells, including thosederived from CD4+ and CD8+ T-cells. In addition, a chemokine responsemay be induced by various white blood or endothelial cells in responseto an administered antigen.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response maybe determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations (e.g., by ELISPOTtechnique), or by measurement of epitope specific T-cells (e.g., by thetetramer technique)(reviewed by McMichael, A. J., and O'Callaghan, C.A., J. Exp. Med. 187(9):1367-1371, 1998; Mcheyzer-Williams, M. G., etal, Immunol. Rev. 150:5-21, 1996; Lalvani, A., et al, J. Exp. Med.186:859-865, 1997).

Thus, an immunological response as used herein may be one thatstimulates the production of CTLs, and/or the production or activationof helper T-cells. The production of chemokines and/or cytokines mayalso be stimulated. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor, cytotoxic,or helper T-cells and/or T-cells directed specifically to an antigen orantigens present in the composition or vaccine of interest. Theseresponses may serve to neutralize infectivity, and/or mediateantibody-complement, or antibody dependent cell cytotoxicity (ADCC) toprovide protection to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

1. Pharmaceutical Compositions

The antigens used in this invention comprise antigens derived from HIV.Such antigens include, for instance, the structural proteins of HIV,such as Env, Gag and Pol. Preferably, the antigens of this inventioncomprise an HIV Env protein, such as gp140. Still more preferably, theantigens of this invention are optimized for immunogenicity andoligomerized, such as Ogp140.

The genes of HIV are located in the central region of the proviral DNAand encode at least nine proteins divided into three major classes: (1)the major structural proteins, Gag, Pol, and Env; (2) the regulatoryproteins, Tat and Rev and (3) the accessory proteins, Vpu, Vpr, Vif, andNef. Many variants are known in the art, including HIV_(SF2),HIV_(IIIb), HIV_(SF2), HIV-1_(SF162), HIV-I_(SF170), HIV_(LAV),HIV_(LA1), HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strainsfrom diverse subtypes (e.g., subtypes, A through G, and O), HIV-2strains and diverse subtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)), andsimian immunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition(W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fieldsand D. M. Knipe, eds. 1991); Virology, 3rd Edition (Fields, B N, D MKnipe, P M Howley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.;for a description of these and other related viruses).

In addition, due to the large immunological variability that is found indifferent geographic regions for the open reading frame of HIV,particular combinations of antigens may be preferred for administrationin particular geographic regions. Briefly, at least eight differentsubtypes of HIV have been identified and, of these, subtype B virusesare more prevalent in North America, Latin America and the Caribbean,Europe, Japan and Australia. Almost every subtype is present insub-Saharan Africa, with subtypes A and D predominating in central andeastern Africa, and subtype C in southern Africa. Subtype C is alsoprevalent in India and it has been recently identified in southernBrazil. Subtype E was initially identified in Thailand, and is alsopresent in the Central African Republic. Subtype F was initiallydescribed in Brazil and in Romania. The most recent subtypes describedare G, found in Russia and Gabon, and subtype H, found in Zaire and inCameroon. Group O viruses have been identified in Cameroon and also inGabon. Thus, as will be evident to one of ordinary skill in the art, itis generally preferred to select an HIV antigen that is appropriate tothe particular HIV subtype that is prevalent in the geographical regionof administration. Subtypes of a particular region may be determined bytwo-dimensional double immunodiffusion or, by sequencing the HIV genome(or fragments thereof) isolated from individuals within that region.Importantly, we have found that antibodies induced by immunizations withOgp140 can neutralize various strains of HIV and therefore can be usedas a prophylactic vaccine in several regions of the world.

As described above, also presented by HIV are various Gag and Envantigens. HIV-1 Gag proteins are involved in many stages of the lifecycle of the virus including, assembly, virion maturation after particlerelease, and early post-entry steps in virus replication. The roles ofHIV-1 Gag proteins are numerous and complex (Freed, E. O. (1998)Virology 251:1-15). For its part, the envelope protein of HIV-1 is aglycoprotein of about 160 kD (gp160). During virus infection of the hostcell, gp160 is cleaved by host cell proteases to form gp120 and theintegral membrane protein, gp41. The gp41 portion is anchored in (andspans) the membrane bilayer of virion, while the gp120 segment protrudesinto the surrounding environment. As there is no covalent attachmentbetween gp120 and gp41, free gp120 is released from the surface ofvirions and infected cells.

The sequences encoding the open reading frame of the ectodomain of theEnv protein (gp140) from the HIV-1_(US4) strain were codon-optimized asdescribed elsewhere [Haas, 1996 #562; zur Megede, 2000 #1451], andconstructed synthetically as a 2.1 kb EcoR1-Xba1 DNA fragment (MidlandReagent Company, Midland, Tex.). This gene cassette contained theprotein-encoding region of the Env protein fused in frame to the humantissue plasminogen activator (tPA) signal sequence as previouslydescribed [Chapman, 1991 #1550]. In order to stabilize the oligomericstructure of the encoded gp140 protein, the DNA sequence was mutated tointroduce an arginine to serine change in the primary protease cleavagesite (REKR) in the Env polypeptide [Earl, 1990 #2906](FIG. 1A). Theresulting Env expression cassette (gp140) was cloned into the EcoR1-Xba1sites of the pCMV3 expression vector for the derivation of stable CHOcell lines. This vector contains the CMV enhancer/promoter elements, anampicillin resistance gene, and sequences encoding a fusion proteincomposed of dihydrofolate reductase (DHFR) and an attenuated neomycinresistance protein.

At least one immunogenic portion of an HIV antigen may be used formucosal immunization. As utilized herein, “immunogenic portion” refersto a portion of the respective antigen that is capable, under theappropriate conditions, of causing an immune response (i.e.,cell-mediated or humoral). The immunogenic portion(s) used forimmunization may be of varying length, although it is generallypreferred that the portions be at least 9 amino acids long and mayinclude the entire antigen. Immunogenicity of a particular sequence isoften difficult to predict, although T cell epitopes may be predictedutilizing computer algorithms such as TSITES (MedImmune, Maryland), inorder to scan coding regions for potential T-helper sites and CTL sites.From this analysis, peptides are synthesized and used as targets in anin vitro cytotoxic assay. Other assays, however, may also be utilized,including, for example, ELISA, or ELISPOT, which detects the presence ofantibodies against the newly introduced vector, as well as assays whichtest for T helper cells, such as gamma-interferon assays, IL-2production assays and proliferation assays.

Immunogenic portions may also be selected by other methods. For example,the HLA A2.1 transgenic mouse has been shown to be useful as a model forhuman T-cell recognition of viral antigens. Briefly, in the influenzaand hepatitis B viral systems, the murine T cell receptor repertoirerecognizes the same antigenic determinants recognized by human T cells.In both systems, the CTL response generated in the HLA A2.1 transgenicmouse is directed toward virtually the same epitope as those recognizedby human CTLs of the HLA A2.1 haplotype (Vitiello et al. (1991) J. Exp.Med. 173:1007-1015; Vitiello et al. (1992) Abstract of Molecular Biologyof Hepatitis B Virus Symposia).

Additional immunogenic portions of the HIV antigens described herein maybe obtained by truncating the coding sequence at various locationsincluding, for example, to include one or more epitopes from the variousdomains of the HIV genome. As noted above, such domains includestructural domains such as Gag, Gag-polymerase, Gag-protease, reversetranscriptase (RT), integrase (IN) and Env. The structural domains areoften further subdivided into polypeptides, for example, p55, p24, p6(Gag); p160, p10, p15, p31, p65 (pol, prot, RT and IN); and gp160, gp120and gp41 (Ems) or Ogp140 as constructed by Chiron Corporation.Additional epitopes of HIV and other sexually transmitted diseases areknown or can be readily determined using methods known in the art. Alsoincluded in the invention are molecular variants of such polypeptides,for example as described in PCT/US99/31245; PCT/US99/31273 andPCT/US99/31272.

Preferably, the antigens of this invention are optimized forimmunogenicity, such as Ogp140.

As used herein, the phrase “optimized” refers to an increase in theimmunogenicity of the proteins, so that they can induce higher quantityand quality of antibodies. Moreover, polynucleotide sequences that canencode Ogp140 can be optimized by codon substitution of wild typesequences. Haas, et al., (Current Biology 6(3):315-324, 1996) suggestedthat selective codon usage by HIV-1 appeared to account for asubstantial fraction of the inefficiency of viral protein synthesis.Andre, et al., (J. Virol. 72(2):1497-1503, 1998) described an increasedimmune response elicited by DNA vaccination employing a synthetic gp120sequence with optimized codon usage. Schneider, et al. (J. Virol.71(7):4892-4903, 1997) discuss inactivation of inhibitory (orinstability) elements (INS) located within the coding sequences of theGag and Gag-protease coding sequences.

The sequences encoding codon-optimized gp140 were cloned into anexpression vector for the evaluation of Env expression in transienttransfection experiments and for protein purification. To facilitate theefficient secretion of recombinant Ogp140 protein, the native HIV signalsequence was replaced by the human tissue-type plasminogen activator(t-PA) signal sequence. The effect of codon optimization on gp140expression was determined by transient transfection of 293 cells withcodon-optimized and native (non-codon optimized) gp140 constructs and,comparison of expression levels by a capture ELISA and immunoblotting.It was shown previously that sequence modification of HIV gagdramatically improved the level of expression [zur Megede, 2000 #1451],similarly, codon optimization also improved the expression of gp140 4 to10 fold compared to the native construct [Haas, 1996 #562]. Using suchsequence-modified constructs we developed stable CHO cell linessecreting 5-15 μg/ml of o-gp140 and gp120. The antigenicity ofoligomeric gp140 with and without a point mutation (R509 to S509) in thegp120/g41 primary protease cleavage site was also evaluated bytransiently transfecting the 293 cells. Expression and structuralcharacterization data indicated that the native form of the HIV-1ectodomain-encoding region did not form gp140 oligomers efficiently(only about 50% of the expressed protein was found to be in oligomericconformation). In contrast, the single R to S mutation in the proteasecleavage site resulted in the expression of stable gp140 protein in itsoligomeric conformation. Therefore, the constructs employing theprotease cleavage site mutation were used for the derivation of stableCHO cell lines for protein production. Cell lines were also derived forthe monomeric US4 gp120. Expression for these stable CHO cell linesranged from 1-15 ug/ml of secreted Env glycoprotein.

The antigens in the immunogenic compositions will typically be in theform of HIV proteins. The proteins can, of course, be prepared byvarious means (e.g. native expression, recombinant expression,purification from cell culture culture, chemical synthesis etc.) and invarious forms (e.g. native, fusions etc.). They are preferably preparedin substantially pure form (i.e. substantially free from other bacterialor host cell proteins).

The invention further includes polynucleotides encoding for either oneor both of the antigens or adjuvants of the invention. Both the antigensand the adjuvants on the invention can be administered in polynucleotideform. The antigens and/or adjuvants of the invention are then expressedin vivo.

The antigens and/or adjuvants of the invention can also be deliveredusing one or more gene vectors, administered via nucleic acidimmunization or the like using standard gene delivery protocols. Methodsfor gene delivery are known in the art. See, e.g., U.S. Pat. Nos.5,399,346, 5,580,859, 5,589,466. The constructs can be delivered eithersubcutaneously, epidermally, intradermally, intramuscularly,intravenous, mucosally (such as nasally, rectally and vaginally),intraperitoneally, orally or combinations thereof. Preferably, theconstructs are delivered mucosally. More preferably, the contructs aredelivered intranasally, intravaginally, or intrarectally.

An exemplary replication-deficient gene delivery vehicle that may beused in the practice of the present invention is any of the alphavirusvectors, described in, for example, U.S. Pat. Nos. 6,342,372; 6,329,201and International Publication WO 01/92552.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. Selected sequences can be insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human GeneTherapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnset al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrieand Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett etal., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human GeneTherapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barret al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering polynucleotides, mucosallyand otherwise, is the enterically administered recombinant poxvirusvaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference) aswell as the vaccinia virus and avian poxviruses. By way of example,vaccinia virus recombinants expressing the genes can be constructed asfollows. The DNA encoding the antigens and/or adjuvants of the inventionis first inserted into an appropriate vector so that it is adjacent to avaccinia promoter and flanking vaccinia DNA sequences, such as thesequence encoding thymidine kinase (TK). This vector is then used totransfect cells that are simultaneously infected with vaccinia.Homologous recombination serves to insert the vaccinia promoter plus thegene encoding the coding sequences of interest into the viral genome.The resulting TK⁻ recombinant can be selected by culturing the cells inthe presence of 5-bromodeoxyuridine and picking viral plaques resistantthereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver genes encoding the antigens and/or adjuvantsof the invention. Recombinant avipox viruses, expressing immunogens frommammalian pathogens, are known to confer protective immunity whenadministered to non-avian species; The use of an avipox vector isparticularly desirable in human and other mammalian species sincemembers of the avipox genus can only productively replicate insusceptible avian species and therefore are not infective in mammaliancells. Methods for producing recombinant avipoxviruses are known in theart and employ genetic recombination, as described above with respect tothe production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429;and WO 92/03545. Picornavirus-derived vectors can also be used. (See,e.g., U.S. Pat. Nos. 5,614,413 and 6,063,384).

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient expression of the coding sequencesof interest (for example, sequences encoding the antigens or adjuvantsof the invention) in a host cell. In this system, cells are firstinfected in vitro with a vaccinia virus recombinant that encodes thebacteriophage T7 RNA polymerase. This polymerase displays exquisitespecificity in that it only transcribes templates bearing T7 promoters.Following infection, cells are transfected with the polynucleotide ofinterest, driven by a T7 promoter. The polymerase expressed in thecytoplasm from the vaccinia virus recombinant transcribes thetransfected DNA into RNA that is then translated into protein by thehost translational machinery. The method provides for high level,transient, cytoplasmic production of large quantities of RNA and itstranslation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad.Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA(1986) 83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virusrecombinants, or to the delivery of genes using other viral vectors, anamplification system can be used that will lead to high level expressionfollowing introduction into host cells. Specifically, a T7 RNApolymerase promoter preceding the coding region for T7 RNA polymerasecan be engineered. Translation of RNA derived from this template willgenerate T7 RNA polymerase that in turn will transcribe more template.Concomitantly, there will be a cDNA whose expression is under thecontrol of the T7 promoter. Thus, some of the T7 RNA polymerasegenerated from translation of the amplification template RNA will leadto transcription of the desired gene. Because some T7 RNA polymerase isrequired to initiate the amplification, T7 RNA polymerase can beintroduced into cells along with the template(s) to prime thetranscription reaction. The polymerase can be introduced as a protein oron a plasmid encoding the RNA polymerase. For a further discussion of T7systems and their use for transforming cells, see, e.g., InternationalPublication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986)189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al.,Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994)22:2114-2120; and U.S. Pat. No. 5,135,855.

Other antigens which may advantageously be included in compositions ofthe invention are:

-   -   a protein antigen from N. meningitidis serogroup B, such as        those in refs. International patent application WO99/24578;        International patent application WO99/36544; International        patent application WO99/57280; International patent application        WO00/22430; Tettelin et al. (2000) Science 287:1809-1815;        International patent application WO96/29412; Pizza et al. (2000)        Science 287:1816-1820 with protein ‘287’ (see below) and        derivatives (e.g. ‘?G287’) being particularly preferred.    -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. International        patent application PCT/IB01/00166; Bjune et al. (1991) Lancet        338(8775):1093-1096; Fukasawa et al. (1999) Vaccine        17:2951-2958; Rosenqvist et al. (1998) Dev. Biol. Stand.        92:323-333; etc.    -   a saccharide antigen from N. meninigitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. i from        serogroup C [see also ref Costantino et al. (1999) Vaccine        17:1251-1263].    -   a saccharide antigen from Streptococcus pneumoniae [e.g.,        Watson (2000) Pediatr Infect Dis J 19:331-332; Rubin (2000)        Pediatr Clin North Am 47:269-285, v.; Jedrzejas (2001) Microbiol        Biol Rev 65:187-207.    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. Bell (2000) Pediatr Infect Dis J 19:1187-1188;        Iwarson (1995) APMIS 103:321-326.    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. Iwarson (1995) APMIS 103:321-326; Gerlich et        al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.]    -   an antigen from hepatitis C virus [e.g. Hsu et al. (1999) Clin        Liver Dis 3:901-915.].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. Gustafsson et al. (1996) N.        Engl. J. Med. 334:349-355; Rappuoli et al (1991) TIBTECH        9:232-238.]    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        3 of Vaccines (1988) eds. Plotkin & Mortimer. ISBN        0-7216-1946-0.] e.g. the CRM₁₉₇ mutant [e.g. Del Guidice et        al. (1998) Molecular Aspects of Medicine 19:1-70.].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of        Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-19464-0.].    -   a saccharide antigen from Haemophilus influenzae B [e.g.        Costantino et al. (1999) Vaccine 17:1251-1263].    -   an antigen from N. gonorrhoeae [e.g. International patent        application WO99/24578; International patent application        WO99/36544; International patent application WO99/57280].    -   an antigen from Chlamydia pneumoniae [e.g. International patent        application PCT/IB01/01445; Kalman et al. (1999) Nature Genetics        21:385-389; Read et al. (2000) Nucleic Acids Res 28:1397-406;        Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527;        International patent application WO99/27105; International        patent application WO00/27994; International patent application        WO00/37494].    -   an antigen from Chlamydia trachomatis [e.g. International patent        application WO99/28475].    -   an antigen from Porphyromonas gingivalis [e.g. Ross et        al. (2001) Vaccine 19:4135-4142].    -   polio anitigen(s) [e.g. Sutter et al. (2000) Pediatr Clin North        Am 47:287-308; Zimmerman & Spann (1999) Am Fam Physician        59:113-118, 125-126] such as IPV or OPV.    -   rabies antigen(s) [e.g. Dreesen (1997) Vaccine 15 Suppl:S2-6]        such as lyophilised inactivated virus [e.g. 77, RabAvert™].    -   measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11        of Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0].    -   influenza antigen(s) [e.g. chapter 19 of [63] Vaccines (1988)        eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.], such as the        haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. McMichael (2000)        Vaccine 19 Suppl 1:S101-107].    -   an antigen from Streptococcus agalactiae (group B streptococcus)        [e.g. Schuchat (1999) Lancet 353(9146):51-6; International        patent application PCT/GB01/04789].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. International patent application PCT/GB01/04789;        Dale (1999) Infect Dis Clin North Am 13:227-43, viii; Ferretti        et al. (2001) PNAS USA 98: 4658-4663].    -   an antigen from Staphlylococcus aureus [e.g. Kuroda et        al. (2001) Lancet 357(9264):1225-1240; see also pages        1218-1219].    -   LTK63 and LTR72 (discussed infra).

Where a saccharide or carbohydrate antigen is included, it is preferablyconjugated to a carrier protein in order to enhance immunogenicity[Ramsay et al. (2001) Lancet 357(9251):195-196. See also: Lindberg(1999) Vaccine 17 Suppl 2:S28-36; Conjugate Vaccines (eds. Cruse et al.)ISBN 3805549326, particularly vol. 10:48-114 etc.]. Preferred carrierproteins are bacterial toxins or toxoids, such as diphtheria, cholera,E. coli heat labile or tetanus toxoids. The CRM₁₉₇ diphtheria toxoid isparticularly preferred. Other suitable carrier proteins include the N.meninigitidis outer membrane protein [European patent application0372501], synthetic peptides [European patent applications 0378881 &0427347], heat shock proteins [International patent applicationWO93/17712], pertussis proteins [International patent applicationWO98/58668; see also EP-04711771, protein D from H. influenzae[International patent application WO00/56360.], toxin A or B from C.difficile [International patent application WO00/61761], etc. Anysuitable conjugation reaction can be used, with any suitable linkerwhere necessary.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens.

The compositions of this invention also include a mucosal adjuvant.

As used herein, the phrase “mucosal adjuvant” refers to an adjuvantsuitable for mucosal delivery. Preferably, the adjuvant is suitable forintranasal, intra-vaginal or intra-rectal delivery.

The phrase “mucosal delivery” refers to delivery or administration of apharmaceutical composition or a vaccine via one or more mucosal routes.Mucosal routes suitable for use in this invention include but are notlimited to oral, intranasal, intragastric, pulmonary, intestinal,rectal, ocular, and vaginal. In a preferred embodiment, the mucosalroute is intranasal.

Where the mucosal delivery is by an intranasal route, the vaccine of theinvention may be in the form of a nasal spray, nasal drops, gel orpowder.

Where the vaccine is for oral route, for instance, it may be in the formof tablets or capsules (optionally enteric-coated), liquid, transgenicplants, etc.

Mucosal adjuvants suitable for use in the invention include but are notlimited to E. coli heat-labile enterotoxins (“LT”), or detoxifiedmutants thereof, such as the K63 or R72 mutants.

E. coli heat-labile toxins are generally ADP-ribosylating bacterialtoxins. These toxins are composed of a monomeric, enymatically active Asubunit which is responsible for ADP-ribosylation of GTP-bindingproteins, and a non-toxic B subunit which binds receptors on the surfaceof the target cell and delivers the A subunit across the cell membrane.The A subunit of wildtype LT is known to increase intracellular cAMPlevels in target cells, which the B subunit is pentameric and is thoughtto bind to GM1 ganglioside receptors. (LT-B is also thought to bind toadditional receptors).

Generally, the wildtype ADP-ribosylating toxins are too toxic for use inhumans. One approach to eliminate or decrease the toxicity of theseproteins is to mutate one or more amino acids in the A subunit.Detoxified ADP-ribosylating toxin mutants are known in the art,including LTK63 and LTR72. See, e.g., WO 98/42375, WO98/18928, and WO97/02348.

As used herein, “detoxified” refers to both completely nontoxic and lowresidual toxic mutants of the toxin in question. Preferably, thedetoxified protein retains a toxicity of less than 0.01% of thenaturally occurring toxin counterpart, more preferably less than 0.001%and even more preferably, less than 0.0001% of the toxicity of thenaturally occurring toxin counterpart. The toxicity may be measured inmouse CHO cells or preferably by evaluation of the morphological changesin T1 cells. In particular, Y1 cells are adrenal tumor epithelial cellswhich become markedly more rounded when treated with a solutioncontaining LT. (Ysamure et al., Cancer Res. (1966) 26:529-535). Thetoxicity of LT is correlated with this morphological transition. Thus,the mutant toxins may be incubated with Y1 cells and the morphologicalchanges of the cells assessed.

The term “toxoid” as used herein generally refers to a geneticallydetoxified toxin.

Regarding the present invention, any detoxified mutant of an E. coliheat labile toxin can be used as a mucosal adjuvant. Such mutantsoptionally comprise one or more amino acid additions, deletions orsubstitutions that result in a molecule having reduced toxicity whileretaining adjuvanticity. If an amino acid is substituted for thewild-type amino acid, such substitutions may be with a naturallyoccurring amino acid or may be with a modified or synthetic amino acid.Substitutions which alter the amphotericity and hydrophilicity whileretaining the steric effect of the substituting amino acid as far aspossible are generally preferred.

The mutants used in the compositions and methods of the invention arepreferably in the form of a holotoxin, comprising the mutated A subunitand the B subunit, which may be oligomeric, as in the wild-typeholotoxin. The B subunit is preferably not mutated. However, it isenvisaged that a mutated A subunit may be used in isolation from the Bsubunit, either in an essentially pure form or complexed with otheragents, which may replace the B subunit and/or its functionalcontribution.

Preferred LT mutants for use in the methods and compositions of theinvention include mutants with one or more of the following mutations: amutation in the A subunit of the serine at position 63, and a mutationin the A subunit of the alanine at position 72, both numbered relativeto the Domenighini reference discussed below. Preferably, the serine atposition 63 is replaced with a lysine and the alanine at position 72 isreplaced with arginine.

For purposes of the present invention, the numbering of LT correspondsto the LT sequences set forth in Domenighini et al., MolecularMicrobiol. (1995) 15:1165-1167. This Domenighini reference isincorporated by reference in its entirety in this application.Specifically, the LT sequences set forth and described in thisDomenighini reference are specifically incorporated herein by referencein their entirety.

Other mucosal adjuvants suitable for use in the invention includecholera toxin (“CT”) or detoxified mutants thereof and microparticles(i.e., a particle of about 100 nm to about 150 μm in diameter, morepreferably about 200 nmn to about 30 μm in diameter, and still morepreferably about 500 nm to about 10 μm in diameter) formed frommaterials that are biodegradable and non-toxic (e.g., a poly(α-hydroxyacid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, apolycaprolactone, etc.).

Preferably, the mucosal adjuvants of the invention are LT mutants suchas the R72 and the K63 mutants.

Microparticles can also be used in the invention as mucosal adjuvants.These are preferably derived from a poly(a-hydroxy acid), in particular,from a poly(lactide) (“PLA”), a copolymer of D,L-lactide and glycolideor glycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or“PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered antigen. The antigen may be entrapped within themicroparticles, or may be adsorbed onto their surfact.

One or more HIV antigens can be used in the vaccine and methods of thisinvention. For instance, Ogp140 antigens can be used with gag antigens.In this embodiment, the Ogp140-containing and the gag-containingmicroparticles may be a mixture of two distinct populations ofmicroparticles, the first containing Ogp140 and the second containinggag. Alternatively, the microparticles may be present as a singlepopulation, with Ogp140 and gag (and any further antigens) distributedevenly.

LT mutants may advantageously be used in combination withmicroparticle-entrapped antigen, resulting in significantly enhancedimmune responses.

Optionally, an immuno-modulatory factor may be added to thepharmaceutical composition.

As used here, an “immuno-modulatory factor” refers to a molecule, forexample a protein that is capable of modulating an immune response.Non-limiting examples of immunomodulatory factors include lymphokines(also known as cytokines), such as IL-6, TGF-β, IL-1, IL-2, IL-3, etc.);and chemokines (e.g., secreted proteins such as macrophage inhibitingfactor). Certain cytokines, for example TRANCE, flt-3L, and a secretedform of CD40L are capable of enhancing the immunostimulatory capacity ofAPCs. Non-limiting examples of cytokines which may be used alone or incombination in the practice of the present invention include,interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3),interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocytemacropliage-colony stimulating factor (GM-CSF), interleukin-1 alpha(IL-1α), interleukin-11 (IL-11), MIP-1γ, leukemia inhibitory factor(LIF), c-kit ligand, thrombopoietin (TPO), CD40 ligand (CD40L), tumornecrosis factor-related activation-induced cytokine (TRANCE) and flt3ligand (flt-3L). Cytokines are comrmercially available from severalvendors such as, for example, Genzyme (Framingham, Mass.), Amgen(Thousand Oaks, Calif.), R&D Systems and Immunex (Seattle, Wash.). Thesequences of many of these molecules are also available, for example,from the GenBank database. It is intended, although not alwaysexplicitly stated, that molecules having similar biological activity aswild-type or purified cytokines (e.g., recombinantly produced or mutantsthereof) and nucleic acid encoding these molecules are intended to beused within the spirit and scope of the invention.

The compositions of the invention will typically be formulated withpharmaceutically acceptable carriers or diluents. As used herein, theterm “pharmaceutically acceptable carrier” refers to a carrier foradministration of the antigens which does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.Suitable carriers may be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Examples of particulate carriers include those derived frompolymethyl methacrylate polymers, as well as microparticles derived frompoly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g.,Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee et al. (1997) JMicroencapsul. 14(2):197-210; O'Hagan et al. (1993) Vaccine 1(2):149-54. Such carriers are well known to those of ordinary skill inthe art. Additionally, these carriers may function as immunostimulatingagents (“adjuvants”). Furthermore, the antigen may be conjugated to abacterial toxoid, such as toxoid from diphtheria, tetanus, cholera,etc., as well as toxins derived from E. coli.

Pharmaceutically acceptable salts can be used therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof acceptable excipients is available in the well-known Remington'sPharmaceutical Sciences.

Pharmaceutically acceptable carriers in therapeutic compositions maycontain liquids such as water, saline, glycerol and ethanol.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

Further, the compositions described herein can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. Preferably, the compositions will include anamount of the antigen sufficient to mount an immunological response. Anappropriate effective amount can be determined by one of skill in theart. Such an amount will fall in a relatively broad range that can bedetermined through routine trials and will generally be an amount on theorder of about 0.1 μg to about 1000 μg, more preferably about 1 μg toabout 300 μg, of particle/antigen.

As set forth above, preferred mucosal adjuvants for use in thisinvention include detoxified mutants of E. coli heat labile toxin (LT),such as LTR72 and LTK63.

Additional adjuvants may also be used in the invention. Such adjuvantsinclude, but are not limited to: (1) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes,etc.); (2) detoxified mutants of a bacterial ADP-ribosylating toxin suchas a cholera toxin (CT), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. WO93/13202; WO92/19265; WO 95/17211; WO98/18928 and WO 01/22993); and (3) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition; oligodeoxy nucleotides containing immunostimulatory CpGmotifs (Cpg); or combinations of any of the above.

2. Methods

The compositions disclosed herein can be administered to a subject togenerate an immune response. Preferably, the composition can be used asa vaccine to treat or prevent HIV infection.

As used herein, “subject” is meant any member of the subphylum chordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The system described above is intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

The compositions will include “immunologically effective amounts” of HIVantigen i.e. amounts sufficient to raise a specific immune response or,more preferably, to treat, reduce, or prevent HIV infection. An immuneresponse can be detected by looking for antibodies to the HIV antigenused (e.g. IgG or IgA) in patient samples (e.g. in blood or serum, inmesenteric lymph nodes, in spleen, in gastric mucosa, and/or in faeces).The precise effective amount for a given patient will depend upon thepatient's age, size, health, the nature and extent of the condition, theprecise composition selected for administration, the patient's taxonomicgroup, the capacity of the patient's immune system to synthesizeantibodies, the degree of protection desired, the formulation of thevaccine, the treating physician's assessment of the medical situation,and other relevant factors. Thus, it is not useful to specify an exacteffective amount in advance, but the amount will fall in a relativelybroad range that can be determined through routine trials, and is withinthe judgement of the clinician. For purposes of the present invention,an effective dose will typically be from about 0.01 mg/kg to 50 mg/kg inthe individual to which it is administered.

3. Techniques and Further Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature eg. SambrookMolecular Cloning, A Laboratory Manual, Second Edition (1989); DNACloning, Volumes I and II (D. N Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller & M. P. Calos eds. 1987, ColdSpring Harbor Laboratory); Mayer & Walker, eds. (1987), ImmunochemicalMethods in Cell and Molecular Biology (Academic Press, London); Scopes,(1987) Protein Purification: Principles and Practice, Second Edition(Springer-Verlag, N.Y.), and Handbook of Experimental Immunology,Volumes I-IV (Weir & Blackwell eds 1986).

The term “comprising” means “including” as well as “consisting”, so acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

A composition containing X is “substantially free” from Y when at least85% by weight of the total X+Y in the composition is X. Preferably, Xcomprises at least ˜90% by weight of the total of X+Y in thecomposition, more preferably at least ˜95% or even 99% by weight.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

EXAMPLE

The following example is offered by way of illustration, and not by wayof limitation.

This example demonstrates the induction of an immune response in rhesusmacaques through mucosal immunization with HIV-1 gag and HIV-1 Ogp140.

Two groups of rhesus macaques were immunized intranasally (IN) with acombination of HIV-1gag p24) and HIV-1 Ogp. Each group contained twoanimals. The animals in Group One were immunized in the presence ofLTK63. The animals in Group Two were immunized in the presence of LTR72.The formulations used for each group are set forth below in Table 1.TABLE 1 Immunization Formulations Ogp140 gag LTK63 LTR72 Group One 300μg 300 μg 100 μg — Group Two 300 μg 300 μg 100 μg — Group Three 300 μg300 μg — 100 μg Group Four 300 μg 300 μg — 100 μg

An antibody mediated response was observed after the course of fiveimmunizations. Serum IgG titers for each animal two weeks post thefourth immunization (2wp4) and two weeks post the fifth immunization(2wp5) are set forth in Tables 2 and 3 below. Table 2 contains theanti-Ogp140 antibody titers. Table 3 contains the anti-gag (p24)antibody titers. Vaginal wash IgA titers for each animal are set forthin Tables 4 and 5 below. Table 4 contains the anti-Ogp antibody titers.Table 5 contains the anti-gag (p24) antibody titers. TABLE 2 SerumAnti-Ogp140 IgG Titers 2wp4 2wp5 Group One Animal One 2,165 4,996 AnimalTwo 21,573 6,528 Group Two Animal One 464 712 Animal Two 7,425 3,665

TABLE 3 Serum Anti-gag (p24) IgG Titers 2wp4 2wp5 Group One Animal One44 1326 Animal Two 553 813 Group Two Animal One 30 164 Animal Two 3534877

TABLE 4 Vaginal Wash Anti-Ogp140 IgA Titers 2wp4 2wp5 Group One AnimalOne 1333 35 Animal Two 154 135 Group Two Animal One 95 217 Animal Two 86335

TABLE 5 Vaginal Wash Anti-gag (p24) IgA Titers 2wp4 2wp5 Group OneAnimal One 16 2 Animal Two 2.5 1.5 Group Two Animal One 17 4 Animal Two15.5 67

1. A composition suitable for mucosal delivery comprising an HIVenvelope antigen and a detoxified mutant A subunit of E. coli heatlabile toxin (LT) selected from one or more of the group consisting ofLTK63 and LTR72.
 2. The composition of claim 1, wherein said heat labiletoxin is LTK63.
 3. The composition of claim 1, wherein said heat labiletoxin is LTR72.
 4. The composition of claim 1, wherein said toxincomprises a holotoxin of said E. coli heat labile toxin.
 5. Thecomposition of claim 1, wherein said envelope protein is selected fromthe group consisting of gp120, gp160 and ogp140.
 6. The composition ofclaim 1, wherein said HIV envelope antigen is optimized forimmunogenicity.
 7. The composition of claim 1, wherein said compositionfurther comprises a second HIV antigen.
 8. The composition of claim 7,wherein said second HIV antigen is optimized for immunogenicity.
 9. Thecomposition of claim 7, wherein said second HIV antigen is selected fromone or more of the group consisting of HIV structural proteins, HIVregulatory proteins, and HIV accessory proteins.
 10. The composition ofclaim 9, wherein said HIV structural protein is selected from the groupconsisting of Gag, Pol and envelope.
 11. The composition of claim 9,wherein said HIV regulatory protein is selected from the groupconsisting of Tat and Rev.
 12. The composition of claim 9, wherein saidHIV accessory protein is selected from the group consisting of Vpu, Vpr,Vif, and Nef.
 13. The composition of claim 10, wherein said second HIVantigen is gag.
 14. The composition of claim 1, wherein said compositionis suitable for intranasal delivery.
 15. The composition of claim 1,wherein said composition is suitable for intra-vaginal delivery.
 16. Thecomposition of claim 1, wherein said composition is suitable forintra-rectal delivery.
 17. A composition suitable for mucosal deliverycomprising a polynucleotide encoding for an HIV envelope protein and adetoxified mutant A subunit of E. coli heat labile toxin (LT) selectedfrom one or more of the group consisting of LTK63 and LTR72.
 18. Acomposition suitable for mucosal delivery comprising an HIV envelopeprotein and a polynucleotide encoding a detoxified mutant A subunit ofE. coli heat labile toxin (LT) selected from one or more of the groupconsisting of LTK63 and LTR72.
 19. A method for raising an immuneresponse in a subject comprising mucosally administering a compositioncomprising an HIV envelope antigen and a detoxified mutant A subunit ofE. coli heat labile toxin (LT) selected from the group consisting ofLTK63 and LTR72.
 20. The method of claim 19, wherein said heat labiletoxin is LTK63.
 21. The method of claim 19, wherein said heat labiletoxin is LTR72.
 22. The method of claim 19, wherein said toxin comprisesa holotoxin of said E. coli heat labile toxin.
 23. The method of claim19, wherein said envelope protein is selected from the group consistingof gp120, gp160 and ogp140.
 24. The method of claim 19, furthercomprising administering a second HIV antigen.
 25. The method of claim24, wherein said second HIV antigen is selected from one or more of thegroup consisting of HIV structural proteins, HIV regulatory proteins,and HIV accessory proteins.
 26. The method of claim 24, wherein saidsecond HIV antigen is gag.
 27. The method of claim 17 wherein saidcomposition is administered intranasally.
 26. The method of claim 19,wherein said composition is administered intravaginally.
 27. The methodof claim 19, wherein said composition is administered intrarectally. 28.A method for raising an immune response in a subject comprisingmucosally administering a composition comprising a polynucleotideencoding an HIV envelope antigen and a detoxified mutant A subunit of E.coli heat labile toxin (LT) selected from the group consisting of LTK63and LTR72.
 29. A method for raising an immune response in a subjectcomprising mucosally administering a composition comprising an HIVenvelope antigen and a polynucleotide encoding a detoxified mutant Asubunit of E. coli heat labile toxin (LT) selected from the groupconsisting of LTK63 and LTR72.